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Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 μm in equivalent spherical diameter, imaged by the 4-Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta, Heterosigma akashiwo, and Prorocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml−1) showed a linear agreement (r2 = 0.73) with the other instruments, but individual comparisons at times had large uncertainty.Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected,provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens-based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments. 

Abstract The ocean's twilight zone (TZ) is a vast, globe-spanning region of the ocean. Home to myriad fishes and invertebrates, mid-water fishes alone may constitute 10 times more biomass than all current ocean wild-caught fisheries combined. Life in the TZ supports ocean food webs and plays a critical role in carbon capture and sequestration. Yet the ecological roles that mesopelagic animals play in the ocean remain enigmatic. This knowledge gap has stymied efforts to determine the effects that extraction of mesopelagic biomass by industrial fisheries, or alterations due to climate shifts, may have on ecosystem services provided by the open ocean. We propose to develop a scalable, distributed observation network to provide sustained interrogation of the TZ in the northwest Atlantic. The network will leverage a “tool-chest” of emerging and enabling technologies including autonomous, unmanned surface and underwater vehicles and swarms of low-cost “smart” floats. Connectivity among in-water assets will allow rapid assimilation of data streams to inform adaptive sampling efforts. The TZ observation network will demonstrate a bold new step towards the goal of continuously observing vast regions of the deep ocean, significantly improving TZ biomass estimates and understanding of the TZ's role in supporting ocean food webs and sequestering carbon. 

The goal of the EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) field campaign is to develop a predictive understanding of the export, fate, and carbon cycle impacts of global ocean net primary production. To accomplish this goal, observations of export flux pathways, plankton community composition, food web processes, and optical, physical, and biogeochemical (BGC) properties are needed over a range of ecosystem states. Here we introduce the first EXPORTS field deployment to Ocean Station Papa in the Northeast Pacific Ocean during summer of 2018, providing context for other papers in this special collection. The experiment was conducted with two ships: a Process Ship, focused on ecological rates, BGC fluxes, temporal changes in food web, and BGC and optical properties, that followed an instrumented Lagrangian float; and a Survey Ship that sampled BGC and optical properties in spatial patterns around the Process Ship. An array of autonomous underwater assets provided measurements over a range of spatial and temporal scales, and partnering programs and remote sensing observations provided additional observational context. The oceanographic setting was typical of late-summer conditions at Ocean Station Papa: a shallow mixed layer, strong vertical and weak horizontal gradients in hydrographic properties, sluggish sub-inertial currents, elevated macronutrient concentrations and low phytoplankton abundances. Although nutrient concentrations were consistent with previous observations, mixed layer chlorophyll was lower than typically observed, resulting in a deeper euphotic zone. Analyses of surface layer temperature and salinity found three distinct surface water types, allowing for diagnosis of whether observed changes were spatial or temporal. The 2018 EXPORTS field deployment is among the most comprehensive biological pump studies ever conducted. A second deployment to the North Atlantic Ocean occurred in spring 2021, which will be followed by focused work on data synthesis and modeling using the entire EXPORTS data set. 

Abstract Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 μm in equivalent spherical diameter, imaged by the 4‐Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta,Heterosigma akashiwo, andProrocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml⁻¹) showed a linear agreement (r²= 0.73) with the other instruments, but individual comparisons at times had large uncertainty. Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected, provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens‐based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments.